Characterization and Functional Analysis of Chalcone Synthase Genes in Highbush Blueberry (Vaccinium corymbosum)

Chalcone synthase (CHS) is the first key enzyme-catalyzing plant flavonoid biosynthesis. Until now, however, the blueberry CHS gene family has not been systematically characterized and studied. In this study, we identified 22 CHS genes that could be further classified into four subfamilies from the highbush blueberry (Vaccinium corymbosum) genome. This classification was well supported by the high nucleotide and protein sequence similarities and similar gene structure and conserved motifs among VcCHS members from the same subfamily. Gene duplication analysis revealed that the expansion of the blueberry CHS gene family was mainly caused by segmental duplications. Promoter analysis revealed that the promoter regions of VcCHSs contained numerous cis-acting elements responsive to light, phytohormone and stress, along with binding sites for 36 different types of transcription factors. Gene expression analysis revealed that Subfamily I VcCHSs highly expressed in fruits at late ripening stages. Through transient overexpression, we found that three VcCHSs (VcCHS13 from subfamily II; VcCHS8 and VcCHS21 from subfamily I) could significantly enhance the anthocyanin accumulation and up-regulate the expression of flavonoid biosynthetic structural genes in blueberry leaves and apple fruits. Notably, the promoting effect of the Subfamily I member VcCHS21 was the best. The promoter of VcCHS21 contains a G-box (CACGTG) and an E-box sequence, as well as a bHLH binding site. A yeast one hybridization (Y1H) assay revealed that three anthocyanin biosynthesis regulatory bHLHs (VcAN1, VcbHLH1-1 and VcbHLH1-2) could specifically bind to the G-box sequence (CACGTG) in the VcCHS21 promoter, indicating that the expression of VcCHS21 was regulated by bHLHs. Our study will be helpful for understanding the characteristics and functions of blueberry CHSs.


Introduction
Chalcone synthase (CHS) is a key enzyme catalyzing the first committed step of flavonoid biosynthesis.Since the first discovery of CHS in parsley (Petroselinum hortense) in 1983 [1], CHSs have been successfully isolated from many plant species, such as Arabidopsis thaliana [2], Ipomoea purpurea [3], Gerbera hybrida [4], Chrysanthemum nankingense [5] and Zea mays [6].Their contributions to the flavonoid metabolism of many plants were widely proved [7,8].For instance, a significant positive correlation between the expression of CHS genes and accumulations of anthocyanin has been reported in kiwifruit [9]; the overexpression of the Freesia hybrid FhCHS1 gene in petunia altered the flower color from white to pink [10]; the virus-induced gene silencing (VIGS) of Actinidia eriantha AeCHS resulted in reduced petal anthocyanin accumulation and bleached red petals [11]; the silencing of the CHS gene of apple (Malus × domestica) resulted in the loss of pigmentation in fruit peel, flower and stem [12]; the silencing of the strawberry (Fragaria × ananassai) FaCHS gene resulted in the blocked accumulation of anthocyanins and the appearance of white areas in strawberries [13].
Evidence has revealed that many transcription factors (TFs) function in regulating flavonoids and anthocyanins biosynthesis by influencing the expression of CHS and some other structural genes.For example, Lilium lancifolium LlMYB3 could bind to the promoter of LlCHS2 to improve its expression, and finally increased the accumulation of anthocyanins [14].The apple (M.domestica) MdBBX22-MdHY5 interaction enhanced the binding ability of MdHY5 to the promoters of MdMYB10 and MdCHS, thus promoting the anthocyanin biosynthesis [15].Glycine max GmMYB176 could recognize and bind to the TAGT(T/A)(A/T) motif sequence within the GmCHS8 promoter to promote the flavonoid biosynthesis in soybean [16].Solanum melongena SmbHLH13 could activate the promoters of SmCHS and SmF3H and positively regulate their expression and flavonoid biosynthesis [17].Both Dendrobium officinale DoMYB5 and DobHLH24 could directly bind to the promoters of DoCHS and DoDFR and regulate their expression.Their co-transformation could significantly upregulate the expression levels of DoCHS and DoDFR [18].
Blueberry (Vaccinium corymbosum L.) is a perennial shrub belonging to the genus Vaccinium of the family Ericaceae.Its fruits are rich with anthocyanins and it is one of the most ideal materials for anthocyanin metabolism research [19][20][21].Plant CHSs usually had multi-gene family characteristics.However, reports on blueberry CHSs were mostly limited to single-gene cloning and quantitative real time PCR and/or comparative transcriptomic analysis-based expression analysis.For example, Lin et al. [22] found that the anthocyanin accumulation and expression of anthocyanin biosynthetic structural genes, including CHS, were high in the peel of blueberry fruits; through transcriptomic analysis of Northern Highbush (V.corymbosum) and Rabbiteye (V.virgatum) blueberry fruits, Günther et al. [23] reported that the interactions between flavonoid biosynthesis structural genes such as CHS and anthocyanin biosynthesis regulatory TFs contributed greatly to the anthocyanin biosynthesis; Han et al. [24] reported that the CHS gene expression was higher in ABA-treated blueberries than in untreated controls at late ripening stages, and their expression levels were significantly positively correlated with anthocyanin content.Up to now, however, systematic analysis of the blueberry CHS gene family has not been reported.In this study, we performed genome-wide identification of blueberry CHS genes, characterized their nucleotide and amino acid sequences and investigated their expression patterns in fruits at different ripening stages based on our previously obtained transcriptome data and quantitative real-time PCR (qRT-PCR) analysis.Meanwhile, blueberry leaf and apple fruit-based transient overexpression experiments were conducted for studying the functions of the three VcCHS genes (VcCHS8, VcCHS13 and VcCHS21) that highly expressed in blueberry fruits at late ripening stages.In addition, a yeast one hybridization (Y1H) assay was applied to study the binding ability of anthocyanin biosynthesis regulatory blueberry bHLHs to the promoter of VcCHS21.Our study will be helpful for understanding the characteristics and functions of CHS genes in blueberry.

Identification and Physiochemical Property Analysis of Blueberry CHSs
In total, 22 VcCHS proteins were identified from highbush blueberry.According to the chromosome location information of the coding genes, they were named VcCHS1~VcCHS22.Bioinformatic analysis results (Table 1) showed that the length in VcCHS proteins ranged from 383 aa (VcCHS17) to 557 aa (VcCHS5), with molecular weight ranging from 42.05 kDa (VcCHS17) to 61.13 kDa (VcCHS5); the isoelectric point ranged from 5.30 (VcCHS19) to 8.26 (VcCHS5).All 22 VcCHS proteins were identified to be hydrophilic proteins, and all blueberry CHSs except VcCHS5, VcCHS10, VcCHS11, VcCHS19 and VcCHS20 were predicted to be stable proteins.Subcellular localization prediction results revealed that all VcCHSs are cytoplasm-localized.Phylogenetic analysis showed that VcCHSs could be divided into four subfamilies (Figure 1A).Subfamily I included eight VcCHSs that clustered with M. domestica MdCHS; Subfamily II contained four VcCHSs that were close to two Solanum lycopersicum SlCHSs, one A. thaliana AtCHS, one Litchi chinensis LcCHS and one Z.mays ZmCHS; Subfamily III contained seven VcCHSs; and Subfamily IV contained three VcCHSs that clustered together with Medicago truncatula MtCHS.
Sequence alignment results showed that the nucleotide similarities among 22 VcCHSs ranged from 37.53% to 99.83% (Figure 1B).The nucleotide similarities among Subfamily I members ranged from 83.69% to 99.23%.The nucleotide similarities among Subfamily II members were all very high, ranging from 98.50% to 99.58%.The nucleotide similarities among Subfamily III members ranged from 59.22% to 99.83%.The nucleotide similarities among the Subfamily IV members were also very high (93.38~99.53%).
The 2000 bp sequences upstream from the start codons of VcCHSs were considered promoters.Promoter similarity analysis revealed that the similarities of the 22 VcCHSs promoters ranged from 24.15% to 99.65% (Figure 1D), among which, the similarities among promoters of Subfamily I, II, III and IV members was 60.04~91.54%(VcCHS7 and VcCHS9 promoters), 94.90~96.97%(VcCHS13 and VcCHS20 promoters), 24.15~99.65%(VcCHS2 and VcCHS17 promoters) and 65.49~81.48%(VcCHS10 and VcCHS11 promoters), respectively.Sequences similarities among VcCHS promoters.The redder the color, the higher the similarity, and the greener the color, the lower the similarity.
By analyzing the gene structures of blueberry CHS family genes, all VcCHS members had introns (Figure 3B).The Subfamily I members contained only one intron.Subfamily II and Subfamily IV members all contained two introns.The number of introns in Subfamily III members ranged from one to three.VcCHS17 contained three introns; VcCHS2, VcCHS5 and VcCHS19 contained two introns; and VcCHS1, VcCHS6 and VcCHS18 contained only one intron.
By analyzing the gene structures of blueberry CHS family genes, all VcCHS members had introns (Figure 3B).The Subfamily I members contained only one intron.Subfamily II and Subfamily IV members all contained two introns.The number of introns in Subfamily III members ranged from one to three.VcCHS17 contained three introns; VcCHS2, VcCHS5 and VcCHS19 contained two introns; and VcCHS1, VcCHS6 and VcCHS18 contained only one intron.

Promoter Analysis Results of Blueberry CHS Genes
Studies have found that CHS promoters contain many light-responsive elements [25].Consistently, by analyzing the cis-acting elements in the VcCHS promoters, 17 kinds of light-responsive elements with a total number of 111 were identified (Figure 4).Notably, 19 (86.4%)VcCHS promoters contained the Box 4 element, 18 (81.8%)promoters contained the GT1-motif element and 17 members (77.3%) had the G-box element on their promoters.The CACGTG sequence is a bHLH binding site in the promoters of CHS genes [26].Further sequence search revealed that this sequence could be found in promoters VcCHS7, VcCHS8, VcCHS9, VcCHS10, VcCHS11 and VcCHS15, which were predicted to be of the G-box.Although the G-box element was not predicted on the VcCHS21 promoter, there was a G-box (CACGTG) in its promoter, suggesting that bHLH could also bind to its promoter.

Promoter Analysis Results of Blueberry CHS Genes
Studies have found that CHS promoters contain many light-responsive elements [25].Consistently, by analyzing the cis-acting elements in the VcCHS promoters, 17 kinds of light-responsive elements with a total number of 111 were identified (Figure 4).Notably, 19 (86.4%)VcCHS promoters contained the Box 4 element, 18 (81.8%)promoters contained the GT1-motif element and 17 members (77.3%) had the G-box element on their promoters.The CACGTG sequence is a bHLH binding site in the promoters of CHS genes [26].Further sequence search revealed that this sequence could be found in promoters VcCHS7, VcCHS8, VcCHS9, VcCHS10, VcCHS11 and VcCHS15, which were predicted to be of the G-box.Although the G-box element was not predicted on the VcCHS21 promoter, there was a G-box (CACGTG) in its promoter, suggesting that bHLH could also bind to its promoter.
The VcCHSs promoters also had many stress-responsive elements.All VcCHSs had high temperature and anoxic specific inducibility-related elements in their promoters.Additionally, 20 (90.9%), 16 (72.7%),11 (50%), 9 (40.9%) and 8 (36.4%) members had defense and stress-related, wounding-related, low temperature-responsive, drought-inducibilityrelated and anoxic specific inducibility-related elements in their promoters, respectively.Additionally, we also identified some growth and development-related cis-acting elements in the promoters of VcCHSs, such as the meristem expression-related element, the zein metabolism regulation-related element and so on.
We further analyzed the transcription factor binding sites in the promoters of VcCHS genes (Figure 5).In total, we identified binding sites for 36 kinds of TFs.In promoters of all VcCHSs, the number of Dof binding sites was the largest (173), followed by MYB (122), and the binding site number of ARR-B was the smallest (only 2).Binding sites for twentyeight kinds of TFs were identified in the VcCHS19 promoter, while in the promoter of VcCHS6, binding sites for only nine types of TFs were identified.Except for VcCHS22, all the other 21 VcCHSs had Dof binding sites in their promoters, and only the VcCHS14 promoter had an ARR-B binding site.Notably, MYB and Trihelix binding sites were identified in promoters of all Subfamily I members, and the bHLH binding site was found in promoters of all Subfamily I members except VcCHS22.We further analyzed the transcription factor binding sites in the promoters of VcCHS genes (Figure 5).In total, we identified binding sites for 36 kinds of TFs.In promoters of all VcCHSs, the number of Dof binding sites was the largest (173), followed by MYB (122), and the binding site number of ARR-B was the smallest (only 2).Binding sites for twenty-eight kinds of TFs were identified in the VcCHS19 promoter, while in the promoter of VcCHS6, binding sites for only nine types of TFs were identified.Except for VcCHS22, all the other 21 VcCHSs had Dof binding sites in their promoters, and only the VcCHS14 promoter had an ARR-B binding site.Notably, MYB and Trihelix binding sites were identified in promoters of all Subfamily I members, and the bHLH binding site was found in promoters of all Subfamily I members except VcCHS22.

Expression Analysis of VcCHS Genes in Blueberry Fruits
Transcriptome data-based gene expression analysis revealed that VcCHS members from Subfamily I had the highest expression levels in blueberry fruits, followed by Subfamily II members, while the Subfamily III and IV members expressed minimally, and some members (VcCHS4, VcCHS10 and VcCHS11) had no expression in fruits at all ripening stages (Figure 6A).Noteworthily, Subfamily I and II members showed significantly higher expression levels in red, purple and blue fruits than in green and pink fruits, suggesting that they might play important roles in the biosynthesis of flavonoids and anthocyanin in blueberry fruits at late ripening stages.
Quantitative real time PCR (qRT-PCR) was further applied to validate the expression of three genes (VcCHS8 and VcCHS21 from Subfamily I, as well as VcCHS13 from Subfamily II) that highly expressed in blueberry fruits at late ripening stages.Consistent with our transcriptome data, the expression levels of the three VcCHSs in fruits at late ripening stages (RF, PF and BF) were significantly higher than in GF and PiF.The expression of VcCHS8 in RF, PF and BF was 8.75-, 15.52-and 15.20-fold of that of GF

Expression Analysis of VcCHS Genes in Blueberry Fruits
Transcriptome data-based gene expression analysis revealed that VcCHS members from Subfamily I had the highest expression levels in blueberry fruits, followed by Subfamily II members, while the Subfamily III and IV members expressed minimally, and some members (VcCHS4, VcCHS10 and VcCHS11) had no expression in fruits at all ripening stages (Figure 6A).Noteworthily, Subfamily I and II members showed significantly higher expression levels in red, purple and blue fruits than in green and pink fruits, suggesting that they might play important roles in the biosynthesis of flavonoids and anthocyanin in blueberry fruits at late ripening stages.
Quantitative real time PCR (qRT-PCR) was further applied to validate the expression of three genes (VcCHS8 and VcCHS21 from Subfamily I, as well as VcCHS13 from Subfamily II) that highly expressed in blueberry fruits at late ripening stages.Consistent with our transcriptome data, the expression levels of the three VcCHSs in fruits at late ripening stages (RF, PF and BF) were significantly higher than in GF and PiF.The expression of VcCHS8 in RF, PF and BF was 8.75-, 15.52-and 15.20-fold of that of GF (Figure 6B), respectively.The expression of VcCHS13 in RF, PF and BF was 1.77-, 2.87-and 3.33-fold of that of GF (Figure 6C), respectively.The expression of VcCHS21 in PiF, RF, PF and BF was significantly higher than that of GF (Figure 6D), accounting for about 27.11-, 285.42-, 365.92-and 370.40fold of GF, respectively.(Figure 6B), respectively.The expression of VcCHS13 in RF, PF and BF was 1.77-, 2.87-and 3.33-fold of that of GF (Figure 6C), respectively.The expression of VcCHS21 in PiF, RF, PF and BF was significantly higher than that of GF (Figure 6D), accounting for about 27.11-, 285.42-, 365.92-and 370.40-fold of GF, respectively.quantitative real time PCR analysis result for VcCHS8, VcCHS13 and VcCHS21, respectively.GF: green fruit; PiF: pink fruit; RF: red fruit; PF: purple fruit; BF: blue fruit.Relative expression levels were calculated using GF as control (1).Different letters above columns represent significant difference at p < 0.05 level.

Effects of Transient Overexpression of VcCHS8, VcCHS13 and VcCHS21 on Flavonoids and Anthocyanin Accumulations in Blueberry Leaves
To study the functions of VcCHSs in flavonoid and anthocyanin biosynthesis, blueberry leaf transient overexpression analysis of VcCHS8, VcCHS13 and VcCHS21 was quantitative real time PCR analysis result for VcCHS8, VcCHS13 and VcCHS21, respectively.GF: green fruit; PiF: pink fruit; RF: red fruit; PF: purple fruit; BF: blue fruit.Relative expression levels were calculated using GF as control (1).Different letters above columns represent significant difference at p < 0.05 level.

Effects of Transient Overexpression of VcCHS8, VcCHS13 and VcCHS21 on Flavonoids and Anthocyanin Accumulations in Blueberry Leaves
To study the functions of VcCHSs in flavonoid and anthocyanin biosynthesis, blueberry leaf transient overexpression analysis of VcCHS8, VcCHS13 and VcCHS21 was performed.At five days post-vacuum inoculation, obvious pigmentation was observed in veins of blueberry leaves overexpressing VcCHS8, VcCHS13 and VcCHS21, but not in empty vector (EV) transformed leaves (Figure 7A).Moreover, the vein of blueberry leaf overexpress-ing the Subfamily I member VcCHS21 was the reddest.By determining the contents of flavonoids and anthocyanin in blueberry leaves (Figure 7B,C), we found that the flavonoid and anthocyanin content in blueberry leaves transiently overexpressing VcCHS8, VcCHS13 and VcCHS21 increased by 78.19%, 31.38% and 115.43%, and by 19.98%, 12.01% and 23.85%, respectively.It is worth noting that the promoting effects of Subfamily I members (VcCHS8 and VcCHS21) were much better than Subfamily II member (VcCHS13), and the promoting effects of VcCHS21 on flavonoids and anthocyanin accumulation were both significantly higher than VcCHS8 and VcCHS13 (p < 0.05).
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 11 of 21 performed.At five days post-vacuum inoculation, obvious pigmentation was observed in veins of blueberry leaves overexpressing VcCHS8, VcCHS13 and VcCHS21, but not in empty vector (EV) transformed leaves (Figure 7A).Moreover, the vein of blueberry leaf overexpressing the Subfamily I member VcCHS21 was the reddest.By determining the contents of flavonoids and anthocyanin in blueberry leaves (Figure 7B and 7C), we found that the flavonoid and anthocyanin content in blueberry leaves transiently overexpressing VcCHS8, VcCHS13 and VcCHS21 increased by 78.19%, 31.38% and 115.43%, and by 19.98%, 12.01% and 23.85%, respectively.It is worth noting that the promoting effects of Subfamily I members (VcCHS8 and VcCHS21) were much better than Subfamily II member (VcCHS13), and the promoting effects of VcCHS21 on flavonoids and anthocyanin accumulation were both significantly higher than VcCHS8 and VcCHS13 (p < 0.05).QRT-PCR was further applied to investigate the influences of VcCHS8, VcCHS13 and VcCHS21 overexpression on the expression of flavonoid biosynthesis-related genes.Results showed that the expression levels of VcCHS8, VcCHI, VcDFR, VcF3H, VcANS and VcUFGT in blueberry leaves overexpressing VcCHS8 were significantly higher than those of the EV (p < 0.05), accounting for approximately 1.56-1.84-,2.01-, 2.23-, 4.28-and 1.43-fold of the EV (p < 0.05), respectively (Figure 7D).The expression levels of VcCHS13, VcCHI, VcF3H and VcANS in blueberry leaves overexpressing VcCHS13 were all significantly higher than those of the EV (p < 0.05), and the expression levels of VcDFR and VcUFGT were slightly higher than the EV, accounting for approximately 2.73-, 1.56-, 2.25-, 1.85-, 1.11-and 1.32-fold of the EV, respectively (Figure 7E).The expression levels of VcCHS21, VcCHI, VcDFR, VcF3H, VcANS and in blueberry leaves overexpressing VcCHS21 were significantly higher than those of the EV (p < 0.05), accounting for approximately 3.59-, 1.86-, 2.59-, 3.67-, 4.13-and 2.32-fold of the EV, respectively (Figure 7F).It is obvious that the gene expression-promoting effect in blueberry leaves of VcCHS21 overexpression was better than the other two VcCHSs.

Effects of Transient Overexpression of VcCHS8, VcCHS13 and VcCHS21 on Flavonoids and Anthocyanin Accumulations in Apple Fruits
Apple fruit transient transformation of VcCHS8, VcCHS13 and VcCHS21 was further conducted.No obvious pigmentation was observed in apple fruit transformed with EV (Figure 8A).However, pigmentations could be observed in apple fruits overexpressing these three VcCHSs, with apple fruit overexpressing VcCHS21 being the reddest.Fruit color index is an important index for fruit pigmentation, and the L*, a* and b* values of apple peels were firstly determined.Results showed that apple fruits overexpressing VcCHS21 had the lowest L* and b* values but the highest a* value (Figure 8B).The transient overexpression of these three VcCHSs also increased the content of flavonoids and anthocyanin in apple fruits (Figure 8C,D).The flavonoid and anthocyanin content in apple fruits transiently overexpressing VcCHS8, VcCHS13 and VcCHS21 increased by 16.83%, 11.88% and 27.33%, and by 35.68%, 18.38% and 87.57%, respectively.It is worth noting that the content of flavonoids and anthocyanin in apple fruits transiently overexpressing VcCHS21 were both significantly higher than fruits overexpressing VcCHS8 or VcCHS13 (p < 0.05).
The influence of VcCHS8, VcCHS13 and VcCHS21 overexpression on the expression of flavonoid and anthocyanin biosynthesis-related genes in apple fruits was further studied using qRT-PCR (Figure 8E-G).The results showed that the overexpression of VcCHS8, VcCHS13 and VcCHS21 all promoted the expression of MdDFR, MdANS and MdUFGT genes in the apple fruits.Interestingly, only the overexpression of VcCHS21 significantly up-regulated the expression of MdCHS (p < 0.05).

Binding Ability of the Anthocyanin Biosynthesis Regulatory bHLH Transcription Factors to the VcCHS21 Promoter
The bHLH transcription factors can activate the expression of flavonoid biosynthetic structural genes by binding to specific regions of their promoters, such as G-box (CACGTG) or E-box (CANNTG) [27].According to the results of gene expression and transient overexpression analysis, it can be concluded that VcCHS21 was a major CHS functioning in the flavonoids and anthocyanin biosynthesis of blueberry.The promoter of VcCHS21 contains a G-box (CACGTG) sequence and bHLH binding site.Therefore, it was hypothesized that the expression of VcCHS21 could be regulated by bHLHs.To verify this hypothesis, the binding activity of four previously identified blueberry anthocyanin biosynthesis regulatory bHLH transcription factors (VcAN1, VcbHLH42-1, VcbHLH1-1 and VcbHLH1-2) to the VcCHS21 promoter was studied using yeast one hybridization (Y1H).The VcCHS21 306 promoter contained both G-box and E-box sequences, while the VcCHS21 129 promoter carried only the G-box sequence.The G-box sequence (CACGTG) was mutated to (AAAATC) in both pCHS21 306 MT and pCHS21 129 MT (Figure 9A).Y1H assay results showed that in the SD/-Ura medium containing 150 ng/mL AbA, the yeast strains separately transformed with pCHS21 306 , pCHS21 129 , pCHS21 306 MT and pCHS21 129 MT, and yeast strains co-transformed with pCHS21 306 /pCHS21 129 and VcbHLH42-1 could not grow; however, yeast strains cotransformed with pCHS21 306 /pCHS21 129 and VcAN1/VcbHLH1-1/VcbHLH1-2 grow well.These indicated that VcAN1, VcbHLH1-1 and VcbHLH1-2 could bind to the VcCHS21 promoter.In addition, the yeast strains that co-transformed with pCHS21 306 MT/pCHS21 129 MT and VcAN1/VcbHLH42-1/VcbHLH1-1/VcbHLH1-2 could not grow on the SD/-Leu medium containing 150 ng/mL AbA, indicating that G-box (CACGTG) was the specific binding sequence of VcAN1, VcbHLH1-1 and VcbHLH1-2 on the VcCHS21 promoter (Figure 9B).Relative expression levels were calculated using EV as 1.Different letters above columns represent significant difference at p < 0.05 level.
The influence of VcCHS8, VcCHS13 and VcCHS21 overexpression on the expression of flavonoid and anthocyanin biosynthesis-related genes in apple fruits was further studied using qRT-PCR (Figure 8E-G).The results showed that the overexpression of VcCHS8, VcCHS13 and VcCHS21 all promoted the expression of MdDFR, MdANS and MdUFGT genes in the apple fruits.Interestingly, only the overexpression of VcCHS21 significantly up-regulated the expression of MdCHS (p < 0.05).pCHS21306/pCHS21129 and VcAN1/VcbHLH1-1/VcbHLH1-2 grow well.These indicated that VcAN1, VcbHLH1-1 and VcbHLH1-2 could bind to the VcCHS21 promoter.In addition, the yeast strains that co-transformed with pCHS21306MT/pCHS21129MT and VcAN1/VcbHLH42-1/VcbHLH1-1/VcbHLH1-2 could not grow on the SD/-Leu medium containing 150 ng/mL AbA, indicating that the G-box (CACGTG) was the specific binding sequence of VcAN1, VcbHLH1-1 and VcbHLH1-2 on the VcCHS21 promoter (Figure 9B).

The Expansion of Blueberry CHS Gene Family Is Mainly Caused by Segmental Duplications
The number of CHS members in different plant genomes varies greatly.For example, there is only one CHS gene in the A. thaliana genome [28].However, there are six, eight, and fourteen CHS members in genomes of I. purpurea [3], G. hybrida [4] and Z. mays [6], respectively.In this study, we identified 22 CHS members from the highbush blueberry genome.Gene duplication, an important mechanism for the evolutionary expansion of gene families and acquiring novel genes and new functions, supports organisms adapting to diverse conditions [29].The 16 CHS members in the C. nankingense genome included

The Expansion of Blueberry CHS Gene Family Is Mainly Caused by Segmental Duplications
The number of CHS members in different plant genomes varies greatly.For example, there is only one CHS gene in the A. thaliana genome [28].However, there are six, eight, and fourteen CHS members in genomes of I. purpurea [3], G. hybrida [4] and Z. mays [6], respectively.In this study, we identified 22 CHS members from the highbush blueberry genome.Gene duplication, an important mechanism for the evolutionary expansion of gene families and acquiring novel genes and new functions, supports organisms adapting to diverse conditions [29].The 16 CHS members in the C. nankingense genome included five pairs of segmental duplication genes and one pair of tandem duplication genes, indicating that segmental duplication contributed to the CHS gene family expansion in C. nankingense [5].Consistent with this, by analyzing the gene duplication events occurred in blueberry CHS gene family, 20 pairs of segmental duplication involving 17 CHS members and one tandem duplicate (VcCHS5 and VcCHS6) were found, indicating that segmental and tandem duplications, especially segmental duplication, contributed to the expansion of blueberry CHS gene family.In this present study, the Ka/Ks value of all duplicated VcCHS gene pairs was estimated to be ≤1.0,suggesting that the VcCHS family members survived the strong selection pressure of purification by elimination substitution and high selection pressure by natural selection during the evolutionary process [29,30].Further, purifying selection can generate pseudogenes and/or genes with conserved functions [31,32].Therefore, it can be inferred that the expansion of the CHS gene family contributed to the high flavonoid and anthocyanin characteristics of blueberry fruits.

The Subfamily I CHS Members Are Closely Related to Flavonoid and Anthocyanin Accumulation in Blueberry Fruits
Our study revealed that blueberry CHS genes can be divided into four subfamilies.The expression levels of Subfamily I and II members in blueberry fruits, especially Subfamily I members, were significantly higher than those from other subfamilies.Moreover, their expression levels in red, purple and blue fruits were significantly higher than those of green and pink fruits, suggesting that they played an important role in the biosynthesis of flavonoids and anthocyanin in blueberry fruits at late ripening stages.
Much research has revealed that the stable or transient overexpression of CHS genes could cause significant color changes of plants [33,34].The blueberry fruit-based transient transformation system is unstable [35].In this study, to verify the effects of the overexpression of VcCHS8, VcCHS13 and VcCHS21 genes, blueberry leaf and apple fruit transient transformation systems were applied.The results showed that all their transient overexpression promoted the accumulation of flavonoids and anthocyanin and significantly up-regulated the expression of flavonoid biosynthetic structural genes in both blueberry leaves and apple fruits.Moreover, only the transient overexpression of VcCHS21 significantly up-regulated the expression of MdCHS in apple fruits.Notably, the promoting effects on accumulations of flavonoids and anthocyanin and the expression of flavonoid biosynthetic structural genes of Subfamily I members (VcCHS8 and VcCHS21) were found to be better than the Subfamily II member (VcCHS13), with VcCHS21 being the best.This indicated that the VcCHS members from Subfamily I play a more important role in the flavonoid and anthocyanin biosynthesis in blueberry fruits.

The Expression of the Blueberry CHSs Is Regulated by Transcription Factors
The biosynthesis of flavonoids and anthocyanin and the expression of their corresponding structural genes, including CHS, can be influenced by many internal or external stimuli [36][37][38][39][40][41].In this study, we found 111 light-responsive elements in the promoters of 22 VcCHSs, indicating that their expression can be significantly influenced by light.Moreover, we also identified many phytohormone-responsive, defense and stress-related, wounding-related, low temperature-responsive and drought-inducibility-related elements in their promoters, suggesting that the expression of VcCHSs can be influenced greatly by many phytohormones and environmental factors.
TFs play an important role in regulating the biosynthesis of flavonoids and anthocyanin [42].The expression of anthocyanin biosynthesis structural genes is usually regulated by the MYB-bHLH-WD40 (MBW) complex [43].The regulatory functions of this complex can be achieved by binding to promoters of anthocyanin biosynthesis-related structural genes and regulating their expression [44].In L. lancifolium [14] and S. melongena [45], MYB can bind to the MYB binding site in the promoter region of CHS to promote anthocyanin accumulation.GmMYB176 can specifically bind to the TAGT(T/A)(A/T) sequence on the GmCHS8 promoter to promote flavonoid biosynthesis in soybean [16].In this study, we identified binding sites for 36 transcription factors from the VcCHS promoters.It is worth noting that MYB binding sites exist on all Subfamily I members promoters, and bHLH binding sites exist on all Subfamily I members promoters except VcCHS22.Therefore, it can be inferred that the expression of Subfamily I VcCHSs may be regulated by MYB and bHLH.
bHLH can bind to the G/E-box in the promoter sequence of target genes [27].In Salvia miltiorrhiza, SmbHLH60 could specifically bind to the G-box (CACGTG) in the SmDFR promoter and suppress its expression to reduce anthocyanin biosynthesis [26].Sequence analysis revealed that there was both G-box (CACGTG) and E-box in the VcCHS21 promoter.Y1H results revealed that three blueberry anthocyanin regulatory VcbHLHs (VcAN1, VcbHLH1-1-1 and VcbHLH1-2) could specifically bind to the G-box (CACGTG) on the VcCHS21 promoter.This supports well the regulation of TFs on the expression of VcCHSs.

Identification of the Blueberry CHS Genes
The blueberry genome data were downloaded from the GENOMEDATABASE FOR VACCINIUM (GDV, https://www.vaccinium.org/,accessed on 8 March 2023) [46], and the protein sequence of A. thaliana CHS was downloaded from TAIR (https://www.arabidopsis.org/, accessed on 8 March 2023) and used to query BLASTP against the blueberry protein database under criterion of e-value ≤ 1 × 10 −5 .Meanwhile, the Hidden Markov Model files of CHS domains PF00195 (Clal_sti_synt_C) and PF02797 (Clal_sti_synt_N) downloaded from the Pfam database (http://pfam.xfam.org/,accessed on 10 March 2023) were used to identify CHS proteins from blueberry protein files by using HMMER 3.0 software embedded in TBtools (e-value ≤ 1 × 10 −5 ) [47].Candidate CHSs obtained through these two methods were further subjected to conserved domain confirmation using CDD (https: //www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/, accessed on 10 March 2023), and only proteins containing both Clal_sti_synt_C and Clal_sti_synt_N domains were remained.One exception is that VaccDscaff22-snap-gene-48.42 was also removed because its protein length is more than 1300 aa.

Bioinformatic Analysis of Blueberry CHSs and Their Encoded Proteins
ExPASy-ProtParam tool (https://web.expasy.org/protparam/,accessed on 10 March 2023) was used to predict the amino acid number, protein molecular weight, isoelectric point, grand average of hydropathicity and instability index of blueberry CHS proteins.For the subcellular localization prediction of VcCHSs, WoLF PSORT (https://wolfpsort.hgc.jp/,accessed on 10 March 2023) was used [48].

Phylogenetic Analysis of Blueberry CHS Proteins
MEGA11 software (Molecular Evolutionary Genetics Analysis Version 11.0) was applied for the multiple sequence alignments of CHS proteins from V. corymbosum, A. thaliana, Z. mays, Litchi chinensis, Medicago truncatula, Malus domestica and Solanum lycopersicum and for phylogenetic tree construction by using the Neighbor Joining (NJ) method with default parameters (bootstrap = 1000).EvolView (https://www.evolgenius.info/evolview/#/,accessed on 10 March 2023) was used for figure drawing [49].

Promoter Analysis
TBtools was used to extract the 2000 bp sequences upstream from the start codons (ATG) of VcCHSs and used as promoter sequences.The cis-acting elements and transcription factor binding sites in promoters of VcCHSs were analyzed using PlantCARE biological replications were made for each gene.GAPDH [54] and Actin [55] were used as internal reference genes for blueberry and apple gene expression analysis, respectively.The relative expression levels of these genes in different samples were calculated using the 2 −∆∆CT method.Primers used for qRT-PCR analysis are listed in Table S1.

Conclusions
In this study, the blueberry CHS gene family was systematically identified and analyzed.A total of 22 CHS members that can be further classified into four subfamilies were identified from highbush blueberry.Gene expression analysis revealed that the Subfamily I members are highly expressed in blueberry fruits at late ripening stages.The transient overexpression of VcCHS8, VcCHS13 and VcCHS21 could increase the accumulation of flavonoid anthocyanins in blueberry leaves and apple fruits, and the overexpression of the Subfamily I member VcCHS21 showed the best promoting effect.Additionally, anthocyanin biosynthesis regulatory bHLHs (VcAN1, VcbHLH1-1-1 and VcbHLH1-2) could specifically bind to the G-box (CACGTG) sequence on the VcCHS21 promoter, suggesting that these bHLHs could regulate blueberry flavonoids and anthocyanin biosynthesis by regulating the expression of CHS.

Figure 2 .
Figure 2. Chromosome localization and synteny analysis results of VcCHSs.The red lines represent segmental duplicated VcCHS gene pairs.The gene names in red are tandem duplicated gene pairs.

Figure 2 .
Figure 2. Chromosome localization and synteny analysis results of VcCHSs.The red lines represent segmental duplicated VcCHS gene pairs.The gene names in red are tandem duplicated gene pairs.

Figure 3 .
Figure 3. Conserved motif distributions in blueberry CHS proteins (A) and gene structures of their corresponding genes (B).UTR: untranslated region; CDS: coding sequence; aa: amino acid; bp: base pair; N: the N terminal of protein; C: the C terminal of protein.

Figure 3 .
Figure 3. Conserved motif distributions in blueberry CHS proteins (A) and gene structures of their corresponding genes (B).UTR: untranslated region; CDS: coding sequence; aa: amino acid; bp: base pair; N: the N terminal of protein; C: the C terminal of protein.

Figure 4 .
Figure 4. Predicted cis-acting elements in the promoters of VcCHSs.

Figure 4 .
Figure 4. Predicted cis-acting elements in the promoters of VcCHSs.

Figure 5 .
Figure 5. Predicted transcription factor binding sites in the promoters of VcCHS genes.

Figure 5 .
Figure 5. Predicted transcription factor binding sites in the promoters of VcCHS genes.

Figure 6 .
Figure 6.Gene expression analysis results of VcCHSs.(A): transcriptome data-based gene expression analysis of VcCHSs.For heatmap drawing, log2(FPKM + 1) values were used.The redder the color, the higher the gene's expression; the greener the color, the lower the gene's expression.(B-D):quantitative real time PCR analysis result for VcCHS8, VcCHS13 and VcCHS21, respectively.GF: green fruit; PiF: pink fruit; RF: red fruit; PF: purple fruit; BF: blue fruit.Relative expression levels were calculated using GF as control (1).Different letters above columns represent significant difference at p < 0.05 level.

Figure 6 .
Figure 6.Gene expression analysis results of VcCHSs.(A): transcriptome data-based gene expression analysis of VcCHSs.For heatmap drawing, log 2 (FPKM + 1) values were used.The redder the color, the higher the gene's expression; the greener the color, the lower the gene's expression.(B-D):quantitative real time PCR analysis result for VcCHS8, VcCHS13 and VcCHS21, respectively.GF: green fruit; PiF: pink fruit; RF: red fruit; PF: purple fruit; BF: blue fruit.Relative expression levels were calculated using GF as control (1).Different letters above columns represent significant difference at p < 0.05 level.

Figure 8 .
Figure 8. Effects of the transient overexpression of VcCHS8, VcCHS13 and VcCHS21 on flavonoid biosynthesis in apple fruits.(A): apple fruits overexpressing EV, VcCHS8, VcCHS13 and VcCHS21.Bar = 1 cm.(B): color parameters of apple fruits overexpressing EV, VcCHS8, VcCHS13 and VcCHS21.(C,D): influences of VcCHS8, VcCHS13 and VcCHS21 transient overexpression on flavonoid and anthocyanin contents in apple fruits.(E-G): influences of VcCHS8, VcCHS13 and VcCHS21 transient overexpression on the expression of flavonoid metabolism-related structural genes in apple fruits.Relative expression levels were calculated using EV as 1.Different letters above columns represent significant difference at p < 0.05 level.

Figure 8 .
Figure 8. Effects of the transient overexpression of VcCHS8, VcCHS13 and VcCHS21 on flavonoid biosynthesis in apple fruits.(A): apple fruits overexpressing EV, VcCHS8, VcCHS13 and VcCHS21.Bar = 1 cm.(B): color parameters of apple fruits overexpressing EV, VcCHS8, VcCHS13 and VcCHS21.(C,D): influences of VcCHS8, VcCHS13 and VcCHS21 transient overexpression on flavonoid and anthocyanin contents in apple fruits.(E-G): influences of VcCHS8, VcCHS13 and VcCHS21 transient overexpression on the expression of flavonoid metabolism-related structural genes in apple fruits.Relative expression levels were calculated using EV as 1.Different letters above columns represent significant difference at p < 0.05 level.

Figure 9 .
Figure 9. Y1H assays results for the binding abilities of anthocyanin regulatory bHLHs on the promoter of VcCHS21.(A): schematic diagrams for the cloned and mutated VcCHS21 promoters.The VcCHS21306 promoter contained both G-box and E-box sequences, while the VcCHS21129 promoter carried only the G-box sequence.'CACGTG' was mutated to 'AAAATC' in both pCHS21306MT and pCHS21129MT.(B): Y1H assay results.MT: mutant.Bar = 5 mm.

Figure 9 .
Figure 9. Y1H assays results for the binding abilities of anthocyanin regulatory bHLHs on the promoter of VcCHS21.(A): schematic diagrams for the cloned and mutated VcCHS21 promoters.The VcCHS21 306 promoter contained both G-box and E-box sequences, while the VcCHS21 129 promoter carried only the G-box sequence.'CACGTG' was mutated to 'AAAATC' in both pCHS21 306 MT and pCHS21 129 MT. (B): Y1H assay results.MT: mutant.Bar = 5 mm.

Table 2 .
Gene duplication analysis results of blueberry CHS genes.Mya: million years ago.For abbreviation, 'VaccDscaff' is removed from all gene IDs in this table.